HIV vaccine strategies have not yet succeeded in generating broadly neutralizing antibody responses in humans. A limited number of broadly neutralizing human monoclonal antibodies against HIV have been identified and have been the subject of intensive study. Combinations of such antibodies can protect macaques from infection with SIV/HIV chimeric virus, suggesting that if they could be elicited in uninfected humans through vaccination, they could provide protective efficacy. These antibodies may be rare in infected humans, however. In contrast, HIV-infected individuals who demonstrate substantial breadth of neutralization in serum are not rare. We hypothesize that a polyspecific response develops during infection in a substantial subset of individuals and accounts for neutralization breadth. The major goal of this proposal is to define the polyspecific response to HIV present in sera from HIV+ individuals with broad neutralizing antibody responses, and to compare the neutralizing antibody specificities with those who demonstrate narrow neutralization breadth. To do this, we will first define the relationship between envelope trimer binding, avidity, and breadth of neutralization using a novel VLP-based assay. We will determine if trimer-specific antibody responses contribute substantially to the breadth of neutralization in polyclonal serum through antibody depletion techniques and epitope mapping. The clonality of the neutralizing antibody response will be determined for broad and narrow neutralizers. Serum analysis will be complemented by the analysis of B cell subsets identified by VLP binding, and phenotypes associated with broad vs. narrow neutralization will be identified. Together, these studies will enlighten our understanding of neutralization breadth and contribute to the design of a neutralizing antibody-based vaccine regimen. It is very likely that an effective HIV vaccine will need to generate neutralizing antibody responses that can prevent infection with the types of HIV that are circulating in the community. It has been very difficult to generate such broad neutralizing responses using standard vaccine techniques. In this project, we will study the antibody responses in individuals who are infected with HIV who have evidence of broad neutralizing responses. By understanding in detail the antibody responses present in these HIV-infected individuals, we will gain insights into how we can better design a protective HIV vaccine that can generate similar responses in uninfected persons.